1. Introduction
The global wound dressing market is increasing and is predicted to rise at a compound annual growth rate (CAGR) of 4.16% from 2024 to 2030 [
1]. Currently, researchers are paying continuous attention to exploring advanced wound care technologies and product enhancement and the development of innovative wound dressings to accelerate the healing. Moreover, it is worth noting that some wound dressings may not be suitable for all types and stages of wounds. A wound is defined as any damage to skin integrity due to incisions, burns, scalds, and human lesions (diabetic foot, venous ulcers, etc.) [
2]. In recent years, plant-based extracts like
Glycyrrhiza glabra L.,
Achillea millefolium L.,
Aloe vera L., and
Calendula officinalis L. have been proposed as alternative to medicinal compounds for improving the healing process [
2].
Glycyrrhiza glabra L., a perennial herbaceous plant of the
Fabaceae family, grows spontaneously in Italy and specifically in Calabria and Abruzzo. It is known that licorice root extract contains many biologically active compounds such as triterpene saponins, glycyrrhizin, flavonoids, and isoflavonoids, which are mainly responsible for antioxidant, anti-inflammatory, and antimicrobial activity [
3,
4]. Unfortunately,
Glycyrrhiza glabra L. extract is characterized by sensitivity to oxidation and photolysis, which decreases its biological activity. Generally, to preserve biological properties, the encapsulation of natural extracts into nanosystems is a strategy that has been investigated [
5,
6]. In this regard, for the first time, our research group investigated the encapsulation of
Glycyrrhiza glabra extract (GG root extract) into ufasomes and their use in developing Spanish broom wound dressings by impregnation.
Ufasomes are “unsaturated fatty acid vesicles” that were discovered for the first time by Gebicki and Hicks in 1973 [
7]. Typically, oleic acid and linoleic acid are the main components of ufasomes, and, compared with liposomes, they present many advantages such as good biocompatibility, the easy bioavailability of raw material, and a simple preparation method. To accelerate the healing process, ufasomes have been used in conjunction with textiles, which are still the most used material for the treatment of wounds [
5]. Among available textiles, we proposed Spanish broom dressings thanks to the availability, renewability, and cleaner and more resilient cultivation [
8]. Our previous studies have already investigated the possibility of Spanish broom dressings being used to deliver various bioactive compounds in the treatment of skin wounds. To the best of our knowledge, no investigation has been conducted on GG root extract-loaded ufasomes intended for wound healing.
In this work, ufasomes (UFAs) made up of oleic and linoleic acid and ufasomes based only on oleic acid, which we indicated as aosomes (AOs), were prepared, respectively. Lipid vesicles were characterized for their physico-chemical and functional properties, such as their size, particle size distribution, ζ potential, encapsulation efficiency, stability over time, and ability to release glycyrrhizin (GZ). In addition, lipid vesicle cytotoxicity and cell proliferation activity were evaluated through a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and cell cycle analysis on WS1 fibroblasts, respectively. The vesicles’ biosafety and antioxidant activity were also evaluated. Finally, the loaded vesicles were sunk into Spanish broom wound dressings, and a scratch test was performed to assess the wound-healing properties.
3. Materials and Methods
3.1. Materials
The roots of Glycyrrhiza glabra L. were provided by Caffo 1915 Group Srl (Limbadi, Vibo Valentia, Italy), and vouchers of the dried plant material were deposited in the Department of Pharmacy and Biotechnology, University of Bologna (Via San Donato 19/2, Bologna, Italy). Oleic acid was purchased from FarmaLabor (Canosa di Puglia, Italy). Lipoid H90 (fat-free sunflower lecithin with 90% phosphatidylcholine, non-GMO) was sourced from Lipoid HmbH (Ludwigshafen, Germany). Ethanol and methanol (purity > 99.8%) and all the solvents were purchased from Sigma-Aldrich (Milan, Italy), as well as 2,2-diphenyl-1- picrylhydrazyl radical (DPPH), calcium carbonate, and linoleic acid (purity >97%). Folin-Ciocalteu reagent was sourced from Titolchimica (Pontecchio Polesine, Italy). Spanish broom dressings were provided by Professor Giuseppe Chidichimo of the University of Calabria (Arcavacata di Rende, CS, Italy). Dulbecco’s modified Eagle medium supplemented with 4.5 g/L of D-glucose was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA), as well as 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) reagent. Fetal bovine serum (FBS), trypsin/EDTA (5 g porcine trypsin and 2 g EDTA), L-glutamine, penicillin, and streptomycin were purchased from Euroclone S.p.A. (Milan, Italy). WS1 human fibroblasts were sourced from ATCC (American Type Culture Collection ATCC, Manassas, VA, USA). Tryton X100 was sourced from Sigma-Aldrich (Milan, Italy); Tert-butyl hydroperoxide (TBH) was purchased from Acros-organics (Geel, Belgium); and 2HDCF-DA was purchased from Sigma-Aldrich (Milan, Italy).
3.2. Preparation of GG Root Extract
In total, 15 g of shredded roots was extracted through maceration in the dark for 72 h using 300 mL of H
2O:EtOH 50:50
v/
v. According to Um et al. [
20], this solution is able to extract the highest amount of antioxidant and bioactive compounds. Moreover, ethanol has the ability to extract phenolics and flavonoids, while water facilitates the extraction of phenolic acids and promotes the swelling of the vegetal matrix [
20]. Subsequently, the hydroalcoholic extract was filtered through paper filters, and the remaining powder was extracted twice using 150 mL of the extracting phase, with the powder being shaken for 1 h each time and then filtered. All the extracts were reunited, and then the solvents were evaporated first through rotavapor (Buchi R-300, Buchi Italia s.r.l., Cornaredo (MI), Italy), which removed ethanol. The extract was then frozen at −20 °C for 1 day. Subsequently, it was placed in the freeze-dryer (Christ, Milan, Italy) for 48 h, which ensured the complete removal of water.
3.3. GG Root Extract Phenolic Richness Determined by HPLC
The phenolic richness of the GG root extract was estimated based on its GZ content. To this aim, an HPLC-DAD method was used [
21]. The instrumentation was a JASCO PU-2089 quaternary pump, a Jasco UV/Vis MD 910 PDA detector, and an autosampler Jasco AS-2057 (Jasco, Tokyo, Japan). The analysis was carried out at 35 °C on a Poroshell 120, 2.7 mm (4.6 × 150 mm) C18 column (Agilent Technologies, Santa Clara, CA, USA) with a flow rate of 0.8 mL/min. Elution started with 98% eluent A (2% acetic acid in HPLC-grade water) and 2% eluent B (2% acetic acid in HPLC-grade acetonitrile). Then, the gradient was 95% A in 10 min., to 90% A in 7 min., to 83% A in 6 min., to 70% A in 6 min., to 0% A in 6 min., to 98% A in 2 min. Identification and quantification were accomplished by using standard solutions of GZ (Sigma Aldrich, Milano) at concentrations spanning from 1 mg/L to 240 mg/L and taking into account the UV/VIS spectrum and the retention time of the peak. GZ eluted at 34.95 min and was quantified (mg/mg extract) via the external standard method. Before the analysis, the extracts were diluted 1:4 with eluent A and filtered using PDVF syringe filters (0.45 mm). Analyses were carried out in triplicate.
3.4. Total Phenolic Content, Total Flavonoid Content, and Antioxidant Activity
The total phenolic content (TPC) was determined using the Folin–Ciocalteu reagent. Briefly, 0.2 mL of an aqueous extract solution (1 mg/mL) was added to 1 mL of 1:10 diluted Folin–Ciocalteu’s phenol reagent, followed by the addition of 0.8 mL of sodium carbonate solution (7.5%
w/
v). After 30 min in the dark at 40 ± 1.0 °C, the absorbance at 750 nm was measured using a UV-Vis 1601 spectrophotometer (Shimadzu, Milan, Italy). Distilled water served as a blank. The TPC was estimated from a standard curve of gallic acid (R
2 = 0.998). All measurements were performed in triplicate, and the results were expressed as the gallic acid equivalent in µg/mg of
Glycyrrhiza glabra extract (µgGAE/mg extract). The total flavonoid content (TFC) was determined using the pharmacopeial method, with minor modifications [
6]. Briefly, 0.1 mL of 5% (
w/
v) AlCl
3 solution was added to 0.9 mL of the extract solution (1 mg/mL
w/
v). After incubation at room temperature for 30 min in the dark, the absorbance was measured at 430 nm. The TFC was estimated from a standard curve of quercetin (R
2 = 0.9998) and was expressed as µg of the quercetin equivalent (µg QE)/mg of the extract. The measurements were performed in triplicate. The antioxidant activity (AA) was determined by the 2,20-di-phenyl-1-picrylhydrazyl radical (DPPH) reduction assay, as described by Brand-Williams et al. (1995) [
22], with minor modification. Briefly, a solution (1 mg/mL
w/
v) of the extract as well as ascorbic acid (used as a standard antioxidant compound) was mixed 1:1 with a solution of DPPH (0.1 mM in methanol) at room temperature. The mixtures were kept in the dark for 30 min, and the absorbance was measured at 517 nm. Methanol was used as a blank solution, and DPPH solution was used as the control. The test was carried out in triplicate. The results are expressed as a percentage of inhibition of the DPPH radical according to the following equation: Inhibition% = [(A0 − A)/A0], where A0 was the absorbance of the DPPH control and A was the absorbance of the sample with DPPH.
3.5. Antibacterial Activity of GG Root Extract
The antibacterial activity of the GG root extract was initially evaluated using the agar well diffusion assay, in accordance with the methodology outlined by Sallustio et al. (2024) [
11]. In summary, 50 μL of each serial twofold dilution (ranging from 16.0 to 0.5 mg/mL) was spotted onto aseptically made wells (diameter 6 m) on Tryptone Soy Agar (TSA, Oxoid) medium. The medium had previously been seeded with approximately 10
6 CFU/mL of the target microorganisms, namely,
Escherichia (
E.)
coli DSM11250 (=ATCC 10536),
Enterococcus (
E.)
hirae DSM3320 (=ATCC 10541), and
Staphylococcus (
S.)
aureus DSM799 (=ATCC 6538). After a 24 h incubation period at 37 °C, the appearance of a clear halo surrounding the wells was related to the antimicrobial effect.
Strains sensitive to GG root extract by the agar well diffusion test were subject to the time–kill assay [
11]. Tryptone Soy Broth (TSB, Oxoid) medium supplemented with GG root extract at different concentrations (16, 8, 4, 2, and 1 mg/mL) was inoculated with indicators at around 10
6 CFU/mL. To discriminate whether the effect was bactericidal or bacteriostatic, population levels were assessed at time 0 as well as after 24 and 48 h of incubation at 37 °C by counting on TSA. Every test was carried out in triplicate.
3.6. Encapsulation of GG Root Extract
3.6.1. Preparation of UFAs
GG root extract-loaded UFAs were prepared by solubilizing LipoidH90 (15 mg/mL) in the oily phase composed of oleic acid (30 mg/mL) and linoleic acid (10 mg/mL) at 32 ± 1 °C until complete solubilization. Subsequently, the lyophilized extract of Glycyrrhiza glabra L. (6 mg/mL) was solubilized in ultrapure water, and it was added drop by drop to the oily phase using a 5 mL syringe. The two phases were homogenized with a high-speed homogenizer (Ultra-Turrax T25 Basic, IKA-WERKE, Germany) for 2 min at 9500 rpm. Finally, ultrasonication with a probe (Ultrasonic convertor model n °C L4, serial C8087, Bergamo, Italy) for 5 min, in a bath of ice, was performed. The unloaded UFAs were prepared as the control.
3.6.2. Preparation of AOs
GG root extract-loaded AOs were prepared by solubilizing LipoidH90 (15 mg/mL) in oleic acid (40 mg/mL) at 32 ± 1 °C until complete solubilization. Afterwards, the lyophilized extract of Glycyrrhiza glabra L. (6 mg/mL) was solubilized in ultrapure water, and it was added, drop by drop, to the oily phase using a 5 mL syringe. Two phases were homogenized with a high-speed homogenizer (Ultra-Turrax T25 basic IKA_WERKE) for 2 min at 9500 rpm. Finally, ultrasonication with a probe (Ultrasonic convertor model n°CL4, serial C8087) for 5 min, in a bath of ice, was performed. The unloaded AOs were prepared as the control.
3.6.3. Characterization of Vesicles
Size, PDI, and Zeta Potential Measurement
The prepared nanovesicular systems were characterized by their vesicle size and their polydispersity index (PDI). The UFAs and AOs were diluted (1:1000 v/v) in MilliQ water before the analysis. The size and PDI were measured through Dynamic Light Scattering (90Plus Particle Size Analyzer, Brookhaven Instruments Corp., Holtsville, NY, USA), while the zeta potential measurements were carried out at 25 °C through a Nicomp™ 380 ZLS instrument (Menlo Park, CA, USA) after the same dilution (1:1000 v/v).
EE
To evaluate the amount of GZ encapsulated in the vesicles, the entrapment efficiency (EE) was determined through the dialysis method. Specifically, 1 mL was purified from the non-incorporated components through dialysis (Spectra/Por® membranes: 14 kDa MW cut-off) in water (0.5 L) for 2 h at room temperature, with the distilled water refreshed after 30 min (2 L in total). At the end of the purification process, the GZ content, before and after dialysis, was estimated through HPLC analyses, as described in paragraph 3.3. The nanovesicles were previously treated with a methanol solution (1:4 ratio); afterwards, the EE was calculated as a percentage of GZ after dialysis versus that before dialysis, as in the following formula:
EE% = (GZ% dialyzed sample/GZ% non-dialyzed sample) × 100
Stability Studies
The physical stability of UFAs and AOs was assessed by monitoring the diameter of the nanovesicles and the polydispersity every 7 days for 4 weeks, and then every 30 days for the next 6 months, through Dynamic Light Scattering (DLS) (90Plus Particle Size Analyzer, BTC). All vesicle suspensions were stored in a refrigerator at 4.0 ± 1.0 °C in the dark. For this study, before the measurement of the size and PDI, aliquots of vesicle suspensions were diluted in ultrapure water (1:1000; v/v) at predetermined periods (0, 7, 14, 21, 28, 60, 90, 120, and 150 days).
The accelerated stability of the loaded and unloaded vesicles was performed with the multi-wavelength analytical photocentrifuge LUMiSizer® (L.U.M. GmbH, Berlin, Germany) using STEP-Technology®. A volume of about 1 mL of nanovesicular systems was placed in a specific cuvette (PA110-135XX), subjected to increasing rotor speeds up to 4000 rpm. The evolution of the transmission profiles, trace instability phenomena, and instability indices were analyzed using SEPView® software 6.4.678.6069.
3.6.4. In Vitro Release Studies
The GZ release profiles from lipid vesicles were carried out using a Franz-type static glass diffusion cell (15 mm jacketed cell with a flat-ground joint and clear glass with a 12 mL receptor volume; diffusion surface area = 1.77 cm
2) equipped with a V6A Stirrer (PermeGearInc., Hellertown, PA, USA). Spectra/Por
® membrane (14 kDa MW cut-off) was placed between the receptor and the donor compartments, and 12 mL of a mixture of 3:7 (
v/
v) ethanol/pH 7.4 buffer was used as the receptor medium. The donor compartment was filled with 1 mL of the vesicle suspension. The systems were kept at 32.0 ± 1.0 °C under magnetic stirring (100 rpm/min). At predetermined time points, the samples (0.2 mL) were withdrawn, and the amount of GZ in each aliquot was analyzed by HPLC (as reported in
Section 3.3). The GZ release profiles were performed in triplicate.
3.6.5. Cell Viability Studies
The human fibroblast WS1 cells were grown in DMEM high glucose, supplemented with 10% fetal bovine serum, 2 mM L-Glutamine, 100 units/mL penicillin, and 100 µg/mL streptomycin, at 37 °C in a 5% CO2/95% air humidified atmosphere. To assess the biocompatibility of the nanovesicular systems, an MTT assay was performed. Briefly, WS1 cells were seeded at 7000 cells/well in a 96-well plate and treated for 24 and 48 h with different concentrations of UFAs or AOs (0.15% and 0.31% v/v) or pure extract (same dilution of nanovesicles, 9.3 μg/mL and 18.6 μg/mL). Subsequently, 10 µL of MTT solution (5 mg/mL) was added, and after 4 h of incubation, the media were replaced with 100 µL of isopropanol to solubilize the formazan crystal. Absorbance at 570 nm was measured by means of the multimode plate reader EnSpire (PerkinElmer, Waltham, MA, USA).
3.6.6. Cell Cycle Evaluation by Flow Cytometry
Cell cycle analysis was performed according to Nüsse et al. [
23]. Briefly, 2 × 10
6 cells were centrifuged, and 1 mL of solution I (0.584 g/L NaCl, 1.139 g/L sodium citrate, 10 mg/L RNase, 0.3 mL/L Nonidet P-40) was added to the cell pellet. After 30 min, 1 mL of solution II (100 mg/L propidium iodide (PI), 0.25 M sucrose, 15 g/L citric acid) was added to complete the fixing and staining. The cell suspension was mixed and kept at 4 °C until flow cytometry measurement. Flow cytometric analyses were carried out using a S3 Cell Sorter (Bio-Rad, Hercules, CA, USA) equipped with an Argon laser. Finally, the data were analyzed with FCSalyzer software.
3.6.7. Biosafety
Human blood was provided according to ethical standards, the Declaration of Helsinki, and national and international guidelines from the Immunohematology and Transfusion Medicine Service Bologna Metropolitan Area (Protocol number 0000816 of 23/02/2024). Erythrocytes from human blood were collected by centrifugation at 116 g for 15 min and then diluted to 5% (v/v) with PBS. Briefly, a biological sample (2 mL) was prepared by adding 1 mL of GG root extract (37.2 μg/mL and 18.6 μg/mL), loaded and unloaded UFAs, or AOs diluted in PBS (0.62% and 0.31% v/v) to 1 mL (5%) red blood cell (RBC) suspension in isotonic buffer solution (pH 7.4). Tryton × 100 (0.1% v/v) and phosphate buffer solution were used as positive and negative controls, respectively. The samples were incubated for 60 min at 37 °C and then were centrifuged at 1100× g for 3 min. The absorbance of the supernatant was detected at 542 nm. The Hemolysis rate was calculated according to the following equation:
3.6.8. Intracellular ROS Assay
Reactive oxygen species were detected in intact cells, according to Oparka et al. [
24], with minor modifications. Briefly, WS1 fibroblasts were seeded at a density of 30,000 cell/cm
2 in a 96-well plate and incubated for 24 h to allow for adhesion. To induce oxidative stress, the cells were exposed to 150 μM tert-butyl hydroperoxide (TBH) in PBS for 30 min. Then, the cells were treated for 24 h with GG root extract (18.6 µg/mL), loaded and unloaded UFAs, and AOs (0.31%
v/
v). After the treatment, the cells were incubated with 2 μM H
2DCFDA (Thermo Fisher Scientific, Waltham, MA, USA) for 30 min. After washing, the samples were analyzed under fluorescence microscopy using a Nikon Eclipse TE300 confocal microscope, applying a λ
exc of 488 nm and a λ
em of 515 nm. Finally, the fluorescence intensity of the acquired images was measured using FIJI software [
25].
3.7. Preparation of Spanish Broom Wound Dressing
3.7.1. Impregnation of Wound Dressing with Vesicles
The final wound dressing was prepared using Spanish broom as the supporting material. Specifically, 100 μL of prepared vesicles (as reported in
Section 3.6.1 and
Section 3.6.2), diluted in 0.31%
v/
v PBS, were absorbed onto a 1 × 1 cm
2 piece of Spanish broom for one hour. Then, the dressings were placed in 3 mL of the culture medium and kept for six hours at 32 °C under shaking at 200 rpm. Next, the dressings were removed and the media was centrifuged at 2000 rpm for 10 min and filtered with a 0.45 μm syringe filter. Finally, the conditioned medium was supplemented with 10% fetal bovine serum, 2 mM L-Glutamine, 100 units/mL penicillin, and 100 µg/mL streptomycin.
3.7.2. Wound Healing Assay
The wound healing capacity of the UFAs and AOs was evaluated by a scratch test. The WS1 fibroblasts were seeded in a 24-well plate at a density of 65,000 cells per well and incubated until confluence. The cell monolayers were manually scraped with a p200 pipette tip and then washed twice with PBS to remove cell debris. Next, WS1 fibroblasts were treated with conditioned media (as reported in
Section 3.6.1) with GG root extract, GG root extract-loaded UFAs, GG root extract-loaded AOs, UFAs, and AOs. QPI (quantitative phase imaging) was executed with the Lifecyte
TM imaging system (Phase Focus, Sheffield, UK), and images were acquired every 1 h for 48 h. Finally, the data were analyzed with the Cell Analysis Toolbox software (Phase Focus, Sheffield, UK).
3.8. Statistical Analysis
Every experiment was performed three times, and all results are shown as the mean ± standard deviation (SD). The data from all the experiments were analyzed using the t-test or one-way and two-way ANOVA tests. The GraphPad Prism software, version 8.0 for Windows (GraphPad Software, San Diego, CA, USA), was used for all graphs, computations, and statistical analyses.
4. Conclusions
In this work, new nanovesicles based on unsaturated fatty acids were successfully used to encapsulate Glycyrrhiza glabra extract. Thanks to their suitable physico-chemical properties, Glycyrrhiza glabra extract-loaded UFAs and AOs are good candidates for developing new pharmaceutical products for wound care. In fact, biological studies have demonstrated their biosafety and antioxidant properties, two important requirements for rapid wound closure, avoiding medical consequences. Considering these promising characteristics, we developed a new tool for wound healing made of Spanish broom dressing impregnated with nanoformulations. Additionally, the scratch test demonstrated that Spanish broom dressing impregnated with GG root extract-loaded UFAs accelerated skin wound closure consistently, indicating the efficacy of this innovative medical device for the treatment of wounds. Future studies will focus on the in vivo evaluation of the new wound dressing developed in this work.